TPhreo jeocbt jescutmivme aoryf : Project 1 is to understand the biochemical and cellular mechanisms for terminating endocannabinoid (EC) signaling in the nervous system. The two major ECs, anandamide (AEA) and 2- arachidonoylglycerol (2-AG), are degraded by disfinct enzymatic pathways in vivo. AEA is principally regulated by the integral membrane enzyme fatty acid amide hydrolase (FAAH), which also hydrolyzes several other bioactive fatty acid amides, including the anti-infiammatory lipid A/-palmitoyl ethanolamine, the satiating factor A/-oleoyl ethanolamine, and the TRP channel agonist A/-arachidonoyl taurine. A major challenge in understanding FAAH-regulated fatty acid amide signaling is to discriminate the distinct physiological functions of endogenous FAAH substrates. 2-AG is hydrolyzed by several enzymes in the brain, including the soluble enzyme monoacylglycerol lipase (MAGL) and the integral membrane hydrolases ABHD6 and ABHD12. A major challenge in understanding 2-AG signaling is to determine the unique biochemical and cellular properties of individual 2-AG hydrolytic enzymes. Prior to enzymatic hydrolysis, both AEA and 2-AG may interact with binding proteins responsible for cellular uptake and transport. A major challenge in understanding EC uptake and transport is to identify the putative proteins that mediate these processes. In the previous funding period, we used a combination of synthetic chemistry, enzymology, molecular biology, and functional proteomic/metabolomic methods to enrich our understanding of EC degradative pathways. We have also developed powerful new chemical and genetic tools to probe the function of these pathways. In this renewal application, we plan to use our suite of chemical and genetic tools to gain further mechanistic insights into EC degradative pathways. Specifically, we aim to: 1) characterize mouse models with altered EC degradative pathways, 2) characterize the biochemical and cell biological properties of brain 2-AG hydrolases, and 3) map lipid-protein interactions in EC degradative pathways using chemical proteomic methods. We anticipate that these studies will enhance our understanding of the biochemical pathways that terminate EC signaling in the nervous system and define key components in these pathways that may serve as drug targets for the treatment of a range of human disorders, including pain, depression, and metabolic disorders.